Protein a compositions and methods of use

ABSTRACT

Methods and compositions for modulating an immune response in a subject are provided. Methods include administering to the subject a composition comprising an effective amount of a lymphocyte differentiation factor, e.g., protein A (PA), sufficient to modulate the immune response. Compositions include a lymphocyte differentiation factor, e.g., protein A (PA), in an amount less than 1 μg.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/453,788, filed Apr. 23, 2012, now allowed, which is acontinuation application of U.S. application Ser. No. 12/882,057, filedSep. 14, 2010 now U.S. Pat. No. 8,168,189, which is a continuationapplication of U.S. application Ser. No. 11/559,713, filed Nov. 14, 2006now U.S. Pat. No. 7,807,170, which is a continuation application of U.S.application Ser. No. 10/121,481, filed Apr. 10, 2002 now U.S. Pat. No.7,211,258, all of which applications are expressly incorporated byreference herein in their entirety.

TECHNICAL FIELD

The invention relates to immune response modulation and treating immunedisorders and pathologies associated with or caused by immune disorders.

BACKGROUND

Protein A is a 40,000 Da glycoprotein extracted from the cell wall ofvarious bacteria. Bacteria use PA as a targeting and binding site fortissue attachment. Protein A has a high affinity for the Fc portion ofcertain immunoglobulin classes and even higher affinity for thoseimmunoglobulins once they have bound antigen. This biochemical propertyof PA has been used in a large number of applications. Theseapplications of PA reflect a use of the Fc binding properties of themolecule or PA's ability to stimulate humoral immunity in the absence ofspecific antigen induction (Superantigen applications).

SUMMARY

The invention is based at least in part on a feature(s) of PA that isdistinct from its Fc binding characteristics and Superantigenproperties. This feature confers one or more of the following activitiesin animals: an ability to re-regulate aberrant process(es) and inhibittissue damage or reverse at least a portion of existing tissue damagecaused by the un-regulated process(es); an ability to re-regulateaberrant or undesirable immune process(es).

The invention therefore provides methods for modulating an immuneresponse in a subject. In one embodiment, a method includesadministering to the subject a composition comprising an effectiveamount of a lymphocyte differentiation factor sufficient to modulate theimmune response. In one aspect, the lymphocyte differentiation factorcomprises protein A (PA).

Also provided are methods for treating an immune dysfunction in asubject with or at risk of an immune dysfunction. In one embodiment, amethod includes administering to the subject a composition comprising aneffective amount of protein A (PA) sufficient to treat the immunedysfunction. In one aspect, the immune dysfunction comprises anautoimmune disorder (e.g., rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, psoriatic arthritis, diabetes mellitus,multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupuserythematosis (SLE), autoimmune thyroiditis, atopic dermatitis,eczematous dermatitis, psoriasis, Sjögren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, vaginitis, proctitis, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitisposterior, interstitial lung fibrosis, Hashimoto's thyroiditis,autoimmune polyglandular syndrome, insulin-dependent diabetes mellitus,insulin-resistant diabetes mellitus, immune-mediated infertility,autoimmune Addison's disease, pemphigus vulgaris, pemphigus foliaceus,dermatitis herpetiformis, autoimmune alopecia, Vitiligo, autoimmunehemolytic anemia, autoimmune thrombocytopenic purpura, perniciousanemia, Guillain-Barre syndrome, Stiff-man syndrome, acute rheumaticfever, sympathetic ophthalmia, Goodpasture's syndrome, systemicnecrotizing vasculitis, antiphospholipid syndrome or an allergy). Inanother aspect, the immune dysfunction comprises an immunodeficiency(e.g., severe combined immunodeficiency (SCID) such as recombinaseactivating gene (RAG 1/2) deficiency, adenosine deaminase (ADA)deficiency, interleukin receptor γ chain (γ_(c)) deficiency,Janus-associated kinase 3 (JAK3) deficiency and reticular dysgenesis;primary T cell immunodeficiency such as DiGeorge syndrome, Nudesyndrome, T cell receptor deficiency, MHC class II deficiency, TAP-2deficiency (MHC class I deficiency), ZAP70 tyrosine kinase deficiencyand purine nucleotide phosphorylase (PNP) deficiency; predominantlyantibody deficiencies such as X-linked agammaglobulinemia (Bruton'styrosine kinase deficiency); autosomal recessive agammaglobulinemia suchas Mu heavy chain deficiency; surrogate light chain (γ5/14.1)deficiency; Hyper-IgM syndrome either X-linked (CD40 ligand deficiency)and others; Ig heavy chain gene deletion; IgA deficiency; deficiency ofIgG subclasses (with or without IgA deficiency); common variableimmunodeficiency (CVID); antibody deficiency with normalimmunoglobulins; transient hypogammaglobulinemia of infancy; interferonγ receptor (IFNGR1, IFNGR2) deficiency; interleukin 12 and interleukin12 receptor deficiency; immunodeficiency with thymoma; Wiskott-Aldrichsyndrome (WAS protein deficiency); ataxia telangiectasia (ATMdeficiency); X-linked lymphoproliferative syndrome (SH2D1A/SAPdeficiency); and hyper IgE syndrome). In yet another aspect, the immunedysfunction comprises an immunodeficiency associated with or secondaryto another disease (e.g., chromosomal instability or defective repairsuch as Bloom syndrome, Xeroderma pigmentosum, Fanconi anemia, ICFsyndrome, Nijmegen breakage syndrome and Seckel syndrome; chromosomaldefects such as Down syndrome (Trisomy 21), Turner syndrome andDeletions or rings of chromosome 18 (18p- and 18q-); skeletalabnormalities such as short-limbed skeletal dysplasia (short-limbeddwarfism) and cartilage-hair hypoplasia (metaphyseal chondroplasia);Immunodeficiency associated with generalized growth retardation such asSchimke immuno-osseous dysplasia, Dubowitz syndrome, Kyphomelicdysplasia with SCID, Mulibrey's nannism, Growth retardation, facialanomalies and immunodeficiency and Progeria (Hutchinson-Gilfordsyndrome); immunodeficiency with dermatologic defects such asectrodactyly-ectodermal dysplasia-clefting syndrome, immunodeficiencywith absent thumbs, anosmia and ichthyosis, partial albinism,Dyskeratosis congenita, Netherton syndrome, Anhidrotic ectodermaldysplasia, Papillon-Lefevre syndrome and congenital ichthyosis;hereditary metabolic defects such as acrodermatitis enteropathica,transcobalamin 2 deficiency, type 1 hereditary orotic aciduria,intractable diarrhea, abnormal facies, trichorrhexis andimmunodeficiency, methylmalonic acidemia, biotin dependent carboxylasedeficiency, mannosidosis, glycogen storage disease, type 1b,Chediak-Higashi syndrome; hypercatabolism of immunoglobulin such asfamilial hypercatabolism, intestinal lymphangiectasia; chronicmuco-cutaneous candidiasis; hereditary or congenital hyposplenia orasplenia; and Ivermark syndrome).

Further provided are methods for reducing an inflammatory response in asubject with or at risk of an inflammatory response. In one embodiment,a method includes administering to the subject a composition comprisingan effective amount of protein A (PA) sufficient to reduce aninflammatory response. In one aspect, the inflammatory response ischronic or acute. In another aspect, the inflammatory response is atleast in part mediated by an antibody (e.g., one or moreauto-antibodies) or at least in part mediated by cellular immunity.

Additionally provided are methods for reducing inflammation in asubject. In one embodiment, a method includes administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to reduce the inflammation. In one aspect, the inflammationis chronic or acute. In another aspect, the inflammation is at least inpart antibody or cell mediated. In still another aspect, the treatmentresults in a reduction in severity of a symptom of inflammation (e.g.,swelling, pain, headache, fever, nausea, skeletal joint stiffness, ortissue or cell damage). In yet another aspect, the treatment results ininhibition of antibody production or lymphoid cell proliferation.

Further provided are methods for inhibiting tissue or cell damage in asubject caused by an inflammatory response or inflammation. In oneembodiment, a method includes administering to the subject a compositioncomprising an effective amount of protein A (PA) sufficient to treatinhibiting tissue or cell damage caused by an inflammatory response orinflammation. In one aspect, the tissue or cell damage is caused by achronic or acute inflammatory response or inflammation. In anotheraspect, the inflammatory response or inflammation is at least in partantibody or cell mediated. In yet another aspect, the tissue or celldamage is present in thymus, liver, kidney, spleen, skin, or a skeletaljoint (e.g., knee, ankle, hip, shoulder, wrist, finger, toe, or elbow).In still another aspect, the treatment results in inhibiting orpreventing further tissue or cell damage.

Methods for treating existing tissue or cell damage in a subject causedby an inflammatory response or inflammation are provided. In oneembodiment, a method includes administering to the subject a compositioncomprising an effective amount of protein A (PA) sufficient to treatexisting tissue or cell damage caused by an inflammatory response orinflammation. In one aspect, the existing tissue or cell damage iscaused by a chronic or acute inflammatory response or inflammation. Inanother aspect, the inflammatory response or inflammation is at least inpart antibody or cell mediated. In yet another aspect, the existingtissue or cell damage is present in thymus, liver, kidney, spleen, skin,or a skeletal joint (e.g., knee, ankle, hip, shoulder, wrist, finger,toe, or elbow). In still other aspects, the treatment results inreversing tissue or cell damage or results in inhibiting or preventingfurther tissue or cell damage.

Methods of treating splenomegalia in a subject are also provided. In oneembodiment, a method includes administering to the subject a compositioncomprising an effective amount of protein A (PA) sufficient to treatsplenomegalia.

Methods of inhibiting proliferation or survival of a splenocyte in asubject having or at risk of having undesirable splenocyte proliferationor survival are additionally provided. In one embodiment, a methodincludes administering to the subject a composition comprising aneffective amount of protein A (PA) sufficient to inhibit proliferationor survival of the splenocyte.

Methods of stimulating differentiation or apoptosis of a splenocyte in asubject having or at risk of having undesirable splenocyte proliferationor apoptosis are further provided. In one embodiment, a method includesadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to stimulate differentiation orapoptosis of the splenocyte.

Methods of reducing antibody production by a splenocyte in a subjecthaving or at risk of having undesirable numbers of an antibody areprovided. In one embodiment, a method includes administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to reduce antibody (e.g., auto-antibody) production by asplenocyte.

Methods of reducing numbers of an antibody producing splenocyte in asubject having or at risk of having undesirable numbers of thesplenocytes are also provided. In one embodiment, a method includesadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to reduce the antibody (e.g.,auto-antibody) producing splenocyte.

Methods of reducing natural killer (NK) cell cytotoxicity in a subjecthaving or at risk of having undesirable NK cell cytotoxicity areadditionally provided. In one embodiment, a method includesadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to reduce undesirable NK cellcytotoxicity.

Methods of inhibiting rejection of a transplanted cell, tissue or organin a subject are further provided. In one embodiment, a method includesadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to inhibit rejection of atransplanted cell, tissue or organ (e.g., an allograft or xenograft). Invarious aspects, PA is administered prior to, substantiallycontemporaneously with, or following transplanting the cell, tissue ororgan.

Methods of stimulating differentiation of lymphoid cells are provided.In one embodiment, a method includes contacting one or more lymphoidcells in vitro, ex vivo or in vivo with a composition comprising aneffective amount of protein A (PA) sufficient to stimulatedifferentiation of one or more lymphoid cells. In various aspects, thelymphoid cell is a T or B cell.

The invention is also based at least in part on the low amounts of PArequired to achieve the activities. In particular, PA has theaforementioned activities at low concentrations, typically less thanamounts used for Superantigen applications.

The invention methods therefore may be practiced with PA in amountseffective to elicit one or more of the activities disclosed herein, butwithout substantial superantigen activity, Fc binding activity orsubstantially stimulating humoral immunity. In one embodiment, an amountis a dose of about 1 picogram to about 1 microgram of PA. In anotherembodiment, an amount is a single dose of about 1 picogram to about 1microgram of PA administered intermittently over about 1 to 15 weeks. Inyet another embodiment, an amount is a single dose of about 1 picogramto about 1 microgram administered of PA on alternating days over about 7to 21 days.

Furthermore, the invention provides compositions that elicit one or moreof the activities disclosed herein in unit dosage form. In oneembodiment, a composition comprises a unit dosage form of PA from about0.5-5, 5-10, 10-20, 20-50 or 50-100, 100-500, 100-1000 picograms. Inanother embodiment, a composition comprises a unit dosage form of PAfrom about 1-10, 10-100, 100-500 or about 500-1000 nanograms. In yetanother embodiment, a composition comprises a unit dosage form of PAsufficient to reduce an inflammatory response or inflammation in asubject.

Pharmaceutical compositions are provided that include a unit dosage formof PA (e.g., 0.5-5, 5-10, 10-20, 20-50 or 50-100, 100-500, 100-1000picograms; 1-10, 10-100, 100-500 or about 500-1000 nanograms).Pharmaceutical compositions are provided that include a unit dosage formof PA that elicits one or more of the activities disclosed herein (e.g.,reduces an inflammatory response or inflammation in a subject).

Kits including a unit dosage form of PA (or pharmaceutical compositions)are also provided, such kits optionally further including instructionsfor use in a method of the invention (e.g., reducing an inflammatoryresponse, inflammation or tissue or cell damage caused by aninflammatory response or inflammation in a subject). In one embodiment,a kit includes a plurality of unit dosage forms of PA. In anotherembodiment, a kit further includes a drug (e.g., that reduces aninflammatory response or inflammation).

DESCRIPTION OF DRAWINGS

FIG. 1 shows the weight gain and growth kinetics of A) control untreatednormal mice (C57BL/6J); B) untreated BXSB mice; and C) BXSB mice with PAtreatment.

FIG. 2 shows data indicating that the E-03 dose of PA rectifies thedecrease in CD69−CD4+ observed in BXSB controls at early time points,although later losing this effect at longer time points of chronictreatment.

DETAILED DESCRIPTION

The invention is based at least in part on the characterization of oneor more activities of protein A (PA) that appear to be distinct from itsSuperantigen properties, Fc binding activity or its ability to stimulatehumoral immunity. These distinct PA activities are believed to be atleast in part attributable to PAs ability to re-regulate or normalizeundesirable or aberrant physiological process(es) such as immunedysfunction. PA's ability to re-regulate or normalize physiologicalprocess(es) results in many different beneficial activities including,for example, modulating aberrant or undesirable immune response (e.g.,re-regulating or normalizing), ameliorating or reducing autoimmunity,reducing inflammation or an inflammatory response, inhibiting orreversing at least a portion of tissue damage caused by an un-regulatedprocess(es) such as an undesirable or aberrant immune response.

More particularly, PA efficacy is demonstrated through the use of acollagen induced arthritis (CIA) murine inflammation model. The inducedimmune response to Type II collagen is antibody mediated causing arapidly progressing inflammatory response which can be assessed bymeasuring the inflammation in affected joints and also by applying astandardized clinical assessment for the affected joints (termed“clinical index” or “CI”). The CI assessment involves both swelling andmobility measures. As shown in Example 1, PA at low concentrationsinhibits an acute inflammatory response in the CIA murine model.Histological examination of knee and ankle joints revealed a reductionin tissue damage as well as of immune cell infiltration of the synovium.

PA efficacy is also demonstrated in BXSB animal model, which representsa combined autoimmune deficiency disease having a genetic basis thatresults in early death of the male animals. As shown in Examples 3 to 8,PA at low concentrations modifies many disease characteristics in theBXSB animal, in many cases re-regulating the various manifestations ofthe disease (cellular and histological) towards base-line levels (i.e.,towards normalization). For example, PA inhibits or prevents the earlyonset of the wasting (weight loss); regulates expansion of the spleniccompartment; inhibits over-expression or -activity of humoral immunity;inhibits over-expression or -activity of cellular immunity; modulatesdifferentiation of cells of lymphoid cell lineage; and ameliorates,reduces or reverses tissue damage caused by or associated with thedisease processes. The data further indicates that PA has the same doseresponse pattern as in the CIA model

Thus, PA activities at low concentrations include, for example, one ormore of regulating expansion of the splenic compartment (modulatingproliferation, apoptosis or differentiation), regulating aberrant orundesirable humoral immunity (inhibiting autoantibody production orinhibiting cells that produce autoantibodies), regulating aberrant orundesirable cellular immunity (normalizing TH₁/TH₂ balance, inhibitingcytotoxicity responses), modulating proliferation, apoptosis, ordifferentiation of cells within the lymphoid cell lineage (e.g.,normalizing T cell populations such as increasing numbers of mature Tcells, e.g., CD69−CD4+), inhibiting or reversing cell or tissue damagecaused by undesirable or aberrant immune response (inhibiting orpreventing disease progression, promoting or enhancing disease reversalor tissue regeneration), and normalizing T or B splenocyte numbers ortheir response to one or more mitogens.

PA is therefore useful in treating a subject in need of one or more ofthe aforementioned activities associated with PA. The inventiontherefore provides, inter alia, methods for modulating an immuneresponse (cellular or humoral), methods for treating an undesirable oraberrant immune response (e.g., immune dysfunction) and methods forinhibiting, preventing or reversing a physiological effect caused by orassociated with an immune response in a subject. In one embodiment, amethod includes administering to a subject a composition comprising aneffective amount of a lymphocyte differentiation factor sufficient tomodulate the immune response. In another embodiment, a method includesadministering to a subject a composition comprising an effective amountof PA sufficient to modulate the immune response.

As used herein, the term “modulate” means a detectable change in anactivity or function or effect to which the term is referring. Modulatecan mean any increase, decrease, reduction, inhibition, prevention,stimulation, promotion, enhancement in the activity or function oreffect to which the term refers. For example, modulating an immuneresponse means that activity or function or an effect of the immuneresponse is detectably changed, e.g., an increase, decrease, reduction,inhibition, prevention, stimulation, promotion, or enhancement ofhumoral or cell mediated immunity. Changes in an immune responseindicative of modulation, including, for example, numbers of T and Bcells, proliferation, apoptosis, differentiation, cytotoxicity, antibodyproduction or numbers of antibody producing cells (e.g.,autoantibodies), mitogen responsiveness, inflammation, cell or tissuedamage, or symptoms thereof, can be measured by a variety of methodsdisclosed herein or known in the art. An “effective amount” or“sufficient amount” means an amount needed to achieve the activity oreffect.

As used herein, the terms “re-regulate,” “normalize” and grammaticalvariations thereof mean a shift towards base line levels. A shifttowards base line levels can include, for example, changes in numbers ofcells, differentiation status, antibody production or amounts ofantibody (e.g., autoantibodies in circulation), cytotoxicity or responseto a mitogen. Thus, to re-regulate or normalize numbers of splenocytesin BXSB spleen, for example, means a return towards the number ofsplenocytes typically found in a normal (e.g., disease free) animalspleen, e.g., C57BL/6. Likewise, to re-regulate or normalizeautoantibodies means to reduce the amount of such antibodies to thosemore typically found in a normal (e.g., disease free) animal. Tore-regulate or normalize populations of T cells in BXSB means, forexample, to shift the T cell population towards that typically observedin C57BL/6, e.g., a change from immature to a mature T cell population.

The amount of re-regulation or normalization that can occur can be areturn to at or near baseline levels typical for a normal animal (within5-25% of baseline), but may be less, for example, a detectable shifttowards baseline levels even though the shift does not return the levelsto at or near baseline (e.g., within 25-100% or 25-200% of baseline).The shift will depend on the extent of deviation from baseline in theuntreated state, the amount of PA administered and what is beingreturned to baseline. For example, splenocyte numbers for BXSB are 5 to6-times greater than C57BL/6. A re-regulation or normalization ofsplenocyte numbers for BXSB would therefore mean that splenocyte numberswere reduced in BXSB following treatment. For example, a reduction from5 to 6-times greater than C57BL/6 mice to 1 to 3-times greater thanC57BL/6 mice, or more, such as within about 10-50% of splenocyte numberstypically observed in C57BL/6 mice. Similarly, in BXSB there is a 200%increase of ANA at 5 weeks and a 1000% increase of ANA at 11 weeks. Are-regulation or normalization of auto-antibodies for BXSB wouldtherefore mean that auto-antibody numbers (e.g., ANA) were reducedfollowing treatment. For example, treatment with 0.01 μg PA returnedthese values to at or near baseline (e.g., within 25% of baseline).Thus, autoantibody numbers may decrease from 10-times greater thanC57BL/6 mice to 5 to 8-times greater than C57BL/6 mice or to 1 to5-times greater than C57BL/6 mice, or more, such as within about 10-50%of autoantibody numbers in C57BL/6 mice.

The invention further provides, inter alia, methods for treating animmune dysfunction in a subject with or at risk of an immunedysfunction. In one embodiment, a method includes administering to asubject a composition comprising an effective amount of protein A (PA)sufficient to treat the immune dysfunction. In one aspect, the immunedysfunction comprises an autoimmune disorder.

As used herein, “immune dysfunction” or “immune disorder” means anundesirable immune response, function or activity, that is greater than(e.g., autoimmunity) or less than (e.g., immunodeficiency) desired. Anundesirable immune response, function or activity can be a normalresponse, function or activity. Thus, normal immune responses that arenot considered aberrant so long as they are undesirable are includedwithin the meaning of these terms. An undesirable immune response,function or activity can also be an abnormal response, function oractivity. An abnormal or an aberrant immune response, function oractivity deviates from normal. Immune dysfunction or disorder can beprimarily humoral or cellular in nature, or both, either chronic oracute.

Immune dysfunction or disorders include disorders characterized by manydifferent physiological symptoms or abnormalities. As disclosed herein,BXSB mouse model is an immune disorder characterized by a vast array ofphysiological symptoms and abnormalities which can be treated inaccordance with the invention (see for example, Examples 4 to 9). Theinvention is therefore useful in treating any immune dysfunction ordisorder characterized by many different physiological symptoms andabnormalities including disorders having one or more physiologicalsymptoms or abnormalities similar to BXSB mouse model, or equivalentdisorders in different species. For example, BXSB mouse is characterizedby aberrant splenocyte proliferation, apoptosis or differentiation whichleads to expansion of the splenic compartment and a consequent increasein numbers of immature splenocytes. Thus, although the particular typesof splenocytes whose numbers increase in BXSB mouse may be differentthan those of another species with an immune disorder (e.g., withrespect to their CD markers), the invention is applicable to anydisorder characterized as having undesirable numbers of immaturesplenocytes (caused by excess cell proliferation, survival or failure ofapoptosis) or decreased numbers of mature splenocytes in a subject.

Thus, as the invention is useful for re-regulating or normalizing manyfacets of an immune response, which leads to ameliorating or reducingone or more of the many different symptoms and abnormalities of theimmune disorder, the invention is broadly applicable to disorders thatare different from that which occurs in BXSB mouse. Of course, disorderstreatable in accordance with the invention include those characterizedas having one or more characteristics, symptoms or abnormalities of BXSBeven if less severe than those present in BXSB mouse.

Particular examples of immune disorders to which the invention appliesinclude autoimmune disorders and immunodeficiencies. Autoimmunedisorders are generally characterized as an undesirable or aberrantresponse, activity or function of the immune system. Immunodeficienciesare generally characterized by decreased or insufficient humoral orcell-mediated immune responsiveness or memory, or increased orundesirable tolerance. Such disorders that may be treated in accordancewith the invention include but are not limited to disorders that causecell or tissue/organ damage in the subject.

Thus, the invention additionally provides, inter alia, methods fortreating an autoimmune disorder in a subject with or at risk of anautoimmune disorder. In one embodiment, a method includes administeringto a subject a composition comprising an effective amount of protein A(PA) sufficient to treat the autoimmune disorder. In various aspects,the autoimmune disorder comprises rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, psoriatic arthritis, diabetesmellitus, multiple sclerosis, encephalomyelitis, myasthenia gravis,systemic lupus erythematosis (SLE), autoimmune thyroiditis, atopicdermatitis, eczematous dermatitis, psoriasis, Sjögren's Syndrome,Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior,interstitial lung fibrosis, Hashimoto's thyroiditis, autoimmunepolyglandular syndrome, insulin-dependent diabetes mellitus,insulin-resistant diabetes mellitus, immune-mediated infertility,autoimmune Addison's disease, pemphigus vulgaris, pemphigus foliaceus,dermatitis herpetiformis, autoimmune alopecia, Vitiligo, autoimmunehemolytic anemia, autoimmune thrombocytopenic purpura, perniciousanemia, Guillain-Barre syndrome, Stiff-man syndrome, acute rheumaticfever, sympathetic ophthalmia, Goodpasture's syndrome, systemicnecrotizing vasculitis, antiphospholipid syndrome or an allergy.

The invention additionally provides, inter alia, methods for treatingimmunodeficiency in a subject with or at risk of an immunodeficiency. Inone embodiment, a method includes administering to a subject acomposition comprising an effective amount of protein A (PA) sufficientto treat the immunodeficiency. In various aspects, the immunodeficiencycomprises severe combined immunodeficiency (SCID) such as recombinaseactivating gene (RAG 1/2) deficiency, adenosine deaminase (ADA)deficiency, interleukin receptor γ chain (γ_(c)) deficiency,Janus-associated kinase 3 (JAK3) deficiency and reticular dysgenesis;primary T cell immunodeficiency such as DiGeorge syndrome, Nudesyndrome, T cell receptor deficiency, MHC class II deficiency, TAP-2deficiency (MHC class I deficiency), ZAP70 tyrosine kinase deficiencyand purine nucleotide phosphorylase (PNP) deficiency; predominantlyantibody deficiencies such as X-linked agammaglobulinemia (Bruton'styrosine kinase deficiency); autosomal recessive agammaglobulinemia suchas Mu heavy chain deficiency; surrogate light chain (γ5/14.1)deficiency; Hyper-IgM syndrome either X-linked (CD40 ligand deficiency)and others; Ig heavy chain gene deletion; IgA deficiency; deficiency ofIgG subclasses (with or without IgA deficiency); common variableimmunodeficiency (CVID); antibody deficiency with normalimmunoglobulins; transient hypogammaglobulinemia of infancy; interferonγ receptor (IFNGR1, IFNGR2) deficiency; interleukin 12 and interleukin12 receptor deficiency; immunodeficiency with thymoma; Wiskott-Aldrichsyndrome (WAS protein deficiency); ataxia telangiectasia (ATMdeficiency); X-linked lymphoproliferative syndrome (SH2D1A/SAPdeficiency); and hyper IgE syndrome). In yet another aspect, the immunedysfunction comprises an immunodeficiency associated with or secondaryto another disease (e.g., chromosomal instability or defective repairsuch as Bloom syndrome, Xeroderma pigmentosum, Fanconi anemia, ICFsyndrome, Nijmegen breakage syndrome and Seckel syndrome; chromosomaldefects such as Down syndrome (Trisomy 21), Turner syndrome andDeletions or rings of chromosome 18 (18p- and 18q-); skeletalabnormalities such as short-limbed skeletal dysplasia (short-limbeddwarfism) and cartilage-hair hypoplasia (metaphyseal chondroplasia);Immunodeficiency associated with generalized growth retardation such asSchimke immuno-osseous dysplasia, Dubowitz syndrome, Kyphomelicdysplasia with SCID, Mulibrey's nannism, Growth retardation, facialanomalies and immunodeficiency and Progeria (Hutchinson-Gilfordsyndrome); immunodeficiency with dermatologic defects such asectrodactyly-ectodermal dysplasia-clefting syndrome, immunodeficiencywith absent thumbs, anosmia and ichthyosis, partial albinism,Dyskeratosis congenita, Netherton syndrome, Anhidrotic ectodermaldysplasia, Papillon-Lefevre syndrome and congenital ichthyosis;hereditary metabolic defects such as acrodermatitis enteropathica,transcobalamin 2 deficiency, type 1 hereditary orotic aciduria,intractable diarrhea, abnormal facies, trichorrhexis andimmunodeficiency, methylmalonic acidemia, biotin dependent carboxylasedeficiency, mannosidosis, glycogen storage disease, type 1b,Chediak-Higashi syndrome; hypercatabolism of immunoglobulin such asfamilial hypercatabolism, intestinal lymphangiectasia; chronicmuco-cutaneous candidiasis; hereditary or congenital hyposplenia orasplenia; or Ivermark syndrome.

Additional particular examples of immune dysfunction or disorders towhich the invention applies include an undesirable or aberrantinflammatory response or inflammation. Such disorders may be mediated bycellular or humoral immunity, or a combination of both.

The invention therefore also provides, inter alia, methods for reducingor inhibiting an inflammatory response or inflammation (chronic oracute) in a subject with or at risk of an inflammatory response orinflammation. In one embodiment, a method includes administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to reduce or inhibit an inflammatory response. In anotherembodiment, a method includes administering to the subject a compositioncomprising an effective amount of protein A (PA) sufficient to reduce orinhibit inflammation. In one aspect, the inflammatory response orinflammation is at least in part mediated by an antibody (e.g., one ormore autoantibodies). In another aspect, the inflammatory response orinflammation is at least in part mediated by cellular immunity. Invarious aspects, a method (e.g., treatment) results in a reduction inseverity or frequency of a symptom of an inflammatory response orinflammation. In particular aspects, the symptom includes one or more ofswelling, pain, headache, fever, nausea, skeletal joint stiffness, ortissue or cell damage. In additional particular aspects, a method (e.g.,treatment) results in inhibition of antibody production or lymphoid cellproliferation.

Immune dysfunction, for example, undesirable or aberrant inflammation oran inflammatory response may cause, directly or indirectly, cell ortissue/organ damage, either to multiple cells, tissues or organs, orspecifically to a single cell type, organ or tissue type. For example,as disclosed in the Examples, CIA and BXSB models exhibited damage inmultiple tissues, as evidenced by changes in histology. Tissues thatexhibited damage included knee, ankle, thymus, kidney and liver.Treatment in accordance with the invention resulted in at least apartial reversal of existing tissue damage or a regeneration of normaltissue (see, for example, Tables 9 and 10).

The invention therefore also provides, inter alia, methods for treating,inhibiting and reversing tissue or cell damage, and promoting orenhancing tissue or cell regeneration in a subject caused by immunedysfunction (e.g., an undesirable or aberrant inflammatory response orinflammation). In one embodiment, a method includes administering to asubject a composition comprising an effective amount of protein A (PA)sufficient to treat existing tissue or cell damage caused by immunedysfunction (e.g., an undesirable or aberrant inflammatory response orinflammation). In another embodiment, a method includes administering toa subject a composition comprising an effective amount of protein A (PA)sufficient to inhibit tissue or cell damage (existing or prophylaxis)caused by immune dysfunction (e.g., a chronic or acute undesirable oraberrant inflammatory response or inflammation). In yet anotherembodiment, a method includes administering to a subject a compositioncomprising an effective amount of protein A (PA) sufficient to reverseexisting tissue or cell damage caused by immune dysfunction (e.g., anundesirable or aberrant inflammatory response or inflammation). In stillanother embodiment, a method includes administering to a subject acomposition comprising an effective amount of protein A (PA) sufficientto promote or enhance tissue or cell regeneration caused by immunedysfunction (e.g., an undesirable or aberrant inflammatory response orinflammation). In one aspect, the inflammatory response or inflammationis at least in part mediated by an antibody (e.g., one or moreautoantibodies). In another aspect, the inflammatory response orinflammation is at least in part mediated by cellular immunity. In yetother aspects, the tissue damage is present in thymus, liver, kidney,spleen, skin, or a skeletal joint. In particular aspects, tissue damagein a skeletal joint is present in knee, ankle, hip, shoulder, wrist,finger, toe, or elbow.

Methods of the invention include treatment methods that inhibit orprevent further tissue or cell damage. Thus, the invention also providesmethods of treating existing tissue or cell damage in a subject causedby immune dysfunction (e.g., an undesirable or aberrant inflammatoryresponse or inflammation), as well as inhibiting or preventing furthertissue or cell damage. In one embodiment, a method includesadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to inhibit or prevent further tissueor cell damage caused by immune dysfunction (e.g., an undesirable oraberrant inflammatory response or inflammation). Examples of existingdamage treatable in accordance with the invention include, for example,tissue or organ damage. Exemplary damage as disclosed herein is presentin thymus, liver, kidney, spleen, skin, or a skeletal joint (e.g., kneeor ankle).

Methods of the invention that include treatment of an inflammatoryresponse or inflammation are desired to reduce a symptom orcharacteristic of an inflammatory response or inflammation. At the wholebody level, an inflammatory response or inflammation is generallycharacterized by swelling, pain, headache, fever, nausea, skeletal jointstiffness or lack of mobility, redness or other discoloration. At thecellular level, an inflammatory response or inflammation ischaracterized by one or more of cell infiltration of the region,production of antibodies (e.g., autoantibodies), production ofcytokines, lymphokines, chemokines, interferons and interleukins, growthand maturation (e.g., differentiation factors), cell proliferation,differentiation, accumulation or migration and cell, tissue or organdamage. Thus, treatment will reduce, inhibit or prevent one or more ofsymptoms (severity or frequency of occurrence) or characteristics of aninflammatory response or inflammation.

Methods of the invention also include treating splenomegalia (i.e.,enlarged spleen) in a subject. Such methods include administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to treat splenomegalia. Without being bound by any theory,treating splenomegalia typically stimulates, increases or promotesproliferation or survival of mature lymphocytes (e.g., T or Bsplenocytes), or differentiation from immature to mature cells, orinhibits or decreases proliferation or survival of immature cells to aphysiological status more typical of a normal animal, i.e., an animalthat does not exhibit splenomegalia. Accordingly, methods forstimulating, increasing or promoting proliferation or survival of maturelymphocytes (e.g., T or B splenocytes) or differentiation from immatureto mature lymphocytes (e.g., T or B splenocytes), and inhibiting ordecreasing proliferation or survival of immature lymphocytes (e.g., T orB splenocytes), are provided.

Methods of the invention further include inhibiting, reducing orpreventing antibody production in a subject. In one embodiment, a methodincludes administering to a subject having an undesirable antibody or anaberrant antibody a composition comprising an effective amount ofprotein A (PA) sufficient to reduce antibody production. Autoantibodiesare but one example of an antibody in which it may be desired toinhibit, reduce or prevent its production. Antibody production can beinhibited, reduced or prevented either directly, by causing the cell(e.g., splenocyte) that produces the antibody to reduce antibodyproduction, or indirectly, by reducing numbers of cells (e.g.,splenocytes) that produce the antibody.

Methods of the invention additionally include inhibiting, reducing orpreventing natural killer (NK) cell cytotoxicity in a subject having orat risk of having undesirable NK cell cytotoxicity. In one embodiment, amethod includes administering to a subject a composition comprising aneffective amount of protein A (PA) sufficient to inhibit, reduce orprevent undesirable NK cell cytotoxicity.

Methods of the invention moreover include stimulating, promoting orenhancing differentiation of a lymphoid cell. In one embodiment, amethod includes contacting a lymphoid cell in vitro, ex vivo or in vivowith a composition comprising an effective amount of protein A (PA)sufficient to stimulate, promote or enhance differentiation of alymphoid cell.

The term “contacting” means direct or indirect binding or interactionbetween two or more entities (e.g., between PA and a cell or molecule).Contacting as used herein includes in solution, in solid phase, invitro, in a cell and in vivo.

Assays for detecting an activity of PA include; cellular changes inlymphocyte numbers, proliferation, apoptosis or survival anddifferentiation include trypan blue exclusion (viability); changes incellular CD markers or other molecules (differentiation); amounts ofantibody (e.g., circulating autoantibodies can be measured using ELISAor other antibody detection assays); tissue or organ improvementincluding inhibiting further damage or reversing existing tissue damage(histology, tissue or organ function, or enzyme levels indicative ofimproved function); whole body effects (weight gain or a decrease inweight loss or wasting, improved mobility); and expansion of spleen(histology, numbers of lymphocytes and their differentiation state) asdisclosed herein and further known in the art.

As the invention can be used to inhibit, reduce or prevent anundesirable immune response in a subject, further provided are methodsfor inhibiting, reducing or preventing rejection of a transplanted cell,tissue or organ in a subject (i.e., Host v. Graft disease). In oneembodiment, a method includes administering to a subject a compositioncomprising an effective amount of protein A (PA) sufficient to inhibit,reduce or prevent rejection of a transplanted cell, tissue or organ.Exemplary cells include neural cells. Exemplary tissues include skin,blood vessel, eye and bone marrow. Exemplary organs include heart, lung,liver and kidney. In various aspects, PA is administered prior to,substantially contemporaneously with, or following transplanting thecell, tissue or organ. The transplanted cell, tissue or organ may be anallograft or xenograft.

As used herein, the terms “transplant,” “transplantation” andgrammatical variations thereof mean grafting, implanting, ortransplanting a cell, tissue or organ from one part of the body toanother part, or from one individual/animal to anotherindividual/animal. The term also includes genetically modified cells,tissue and organs, e.g., by ex vivo gene therapy in which thetransformed cells, tissue and organs are obtained or derived from theperson who then receives the transplant, or from a differentperson/animal.

Methods and compositions of the invention may be used in vitro, ex vivoor in vivo. Compositions can be administered as a single or multipledosage form, on consecutive or alternating days or intermittently. Forexample, single or multiple dosage forms can be administered onalternating days or intermittently, over about 7 to 45 days or overabout 1 to 15 weeks. In one embodiment, a composition is administered asa single dose on alternating days for between 3 and 5 weeks.

Treatment usually results in an improvement in the subject's condition,that is a change beneficial to the subject, tissue or cell or cellpopulation in the subject that is detectable. Thus, treatment can resultin inhibiting, reducing or preventing a progression or worsening of thecondition or disorder or symptoms, or further deterioration or onset ofone or more additional symptoms of the condition or disorder. Thus, asuccessful treatment outcome leads to a “therapeutic effect,” orinhibiting, reducing or preventing the severity or frequency of symptomsor underlying causes of a disorder or condition in the subject.Stabilizing a disorder or condition is also a successful treatmentoutcome. Therefore, treatment can reduce or prevent severity orfrequency of one or more symptoms of the condition or disorder, inhibitprogression or worsening of the condition or disorder, and in someinstances, reverse the condition or disorder. Thus, in the case of animmune disorder, for example, treatment can lead to an improvement of ahistopathological change caused by or associated with the immunedisorder, for example, preventing further or reducing or regeneratingskeletal joint infiltration or tissue destruction, or thymus, kidney,liver, spleen, or skin tissue infiltration or tissue destruction.

Treatment also includes affecting the underlying causes of the conditionor disorder or symptoms thereof. Thus, in the case of an immunedisorder, for example, re-regulating or normalizing absolute numbers oflymphocytes (e.g., splenocytes) or numbers of mature lymphocytes towardsnormal baseline is considered a successful treatment outcome. Similarly,a reduction of circulating antibodies (e.g., autoantibodies) towardsnormal baseline is considered a successful treatment outcome.

The term “ameliorate” means a detectable improvement in the subject'soverall condition. A detectable improvement includes a subjectivereduction in the severity or frequency of symptoms caused by orassociated with the disorder or condition, an improvement in theunderlying causes of the disorder or condition, or a reversal of thedisorder or condition, which is detectable using an assay.

Methods of the invention may be practiced prior to (i.e. prophylaxis) orafter symptoms begin, before or after symptoms or the disorder develop(e.g., before cell, tissue or organ transplantation). Administering acomposition prior to or immediately following development of symptomsmay decrease the severity or frequency of the symptoms in the subject.In addition, administering a composition prior to or immediatelyfollowing development of symptoms may decrease or prevent damage tocells, tissues and organs that occurs, for example, during immunedysfunction (e.g., autoimmunity).

The term “subject” refers to animals, typically mammalian animals, suchas a non-human primate (gorillas, chimpanzees, orangutans, macaques,gibbons), a domestic animal (dogs and cats), a farm animal (horses,cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit,guinea pig) and humans. Human subjects include adults and children.Human subjects include those having or at risk of having immunedysfunction. At risk subjects can be identified through geneticscreening. Particular examples of genetically linked immune disordersthat may be identified include X-linked severe combinedimmunodeficiency, Adenosine deaminase deficiency, DiGeorge Anomaly,Ataxia-telangiectasia, Wiscott-Aldrich Syndrome, Leukocyte adhesiondeficiency, and Myotonic dystrophy. These and other disorders aredetectable through fetal blood or amniotic cells, or through adulttissue samples as described in Samter's Immunologic Diseases; M M Frank,K F Austen, H N Claman, and E R Unanue editors; Little, Brown andCompany. Reviewing family history may be used to detect inheritancepatterns or an increased risk (predisposition) of developing thedisorder (e.g., autoimmunity or immunodeficiency). At risk subjects mayalso be identified by screening for a specific characteristic, such asthe presence of undesirable or aberrant populations of lymphocytes(e.g., splenocytes) or autoantibodies. At risk subjects include those inneed of a cell, tissue or organ transplant. Subjects further includedisease model animals (e.g., such as mice and non-human primates) fortesting in vivo efficacy of the compositions of the invention (e.g.,CIA, BXSB, EAE and SCID murine models).

The invention is practiced with compounds known as “lymphocytedifferentiation factors,” which are molecules capable of regulating ormodulating cell signaling or response to signaling, which in turn canre-regulate, normalize or modulate cell behavior of the cell itself,other cells or processes in which the cells participate (e.g., immunesystem function). As set forth herein, a specific example of alymphocyte differentiation factor is PA. Lymphocyte differentiationfactors can be used in accordance with the invention in low amounts asset forth herein for PA.

The invention is also based at least in part on the low amounts of PAthat can produce one or more of activities disclosed herein. Forexample, PA at 1×10⁻⁵ μg (1×10⁻¹¹ G) per dose administered onalternating days (M/W/F) starting at the time of secondary antigeninduction regulated expression and/or progression of the ensuinginflammatory response and regulated expression and/or reversed thetissue damage caused by the inflammatory response. However, at thisamount of PA, there was no substantial superantigen activity orstimulation of humoral immunity.

The invention therefore also provides compositions, including PA in anamount that is able to produce one or more of the activities associatedwith PA, without producing substantial superantigen activity,substantial stimulation of humoral immunity or is substantiallyindependent of Fc binding. Activities of PA in such amounts include, forexample, re-regulating or normalizing aberrant or undesirable humoral orcellular immune response (modulating lymphocyte proliferation, apoptosisor differentiation), inhibiting, reversing, ameliorating or reducingautoimmunity, inflammation or an inflammatory response, or at least aportion of tissue damage caused by an undesirable or aberrant immuneresponse (inhibiting or preventing disease progression, promoting orenhancing disease reversal or tissue regeneration), and normalizing T orB splenocyte numbers or their response pattern to one or more mitogens.

Thus, in one embodiment, a composition includes PA in an amountsufficient to modulate lymphocyte proliferation, apoptosis ordifferentiation. In another embodiment, a composition includes an amountof PA in an amount sufficient to inhibit, reverse, ameliorate or reduceautoimmunity, inflammation or an inflammatory response. In yet anotherembodiment, a composition includes an amount of PA in an amountsufficient to inhibit, reverse, ameliorate or reduce at least a portionof cell, tissue or organ damage caused by an undesirable or aberrantimmune response. In still other embodiments, a composition includes anamount of PA in an amount sufficient to re-regulate or normalize T or Bsplenocyte numbers or their response to one or more mitogens. In oneaspect, the amount of PA is less than 1 μg. In another aspect, theamount of PA is less than 1 μg but greater than 0.01 picograms (pG). Inyet another aspect, the amount of PA is less than 0.5 to 0.1 μg butgreater than 0.1 pG. In still another aspect, the amount of PA is lessthan 0.1 to 0.01 μg but greater than 1 pG. In additional aspects, theamount of PA is less than 1 to 0.1 μg but greater than 1 pG; less than0.1 to 0.01 μg but greater than 1 pG; less than 0.01 to 0.001 μg butgreater than 1 pG; less than 1 to 0.5 ng but greater than 1 pG; lessthan 500 to 250 pG but greater than 1 pG; less than 250 to 50 pG butgreater than 5 pG; and less than 50 to 25 pG but greater than 5 pG,e.g., 20, 15, or about 10 pG. In yet additional aspects, the amount ofPA does not produce substantial superantigen activity, substantialstimulation of humoral immunity or is substantially independent of Fcbinding.

As used herein, the phrases “without substantial” or “substantiallyindependent,” when used in reference to superantigen activity,stimulation of humoral immunity or Fc binding of PA, means that thecharacteristic referred to does not contribute significantly to theobserved activity of PA at that amount. Thus, an amount of PA that doesnot produce substantial superantigen activity means that PA'ssuperantigen activity does not contribute significantly to the activityof that amount of PA. Similarly, an amount of PA that does not producesubstantial stimulation of humoral immunity may produce a small amountof humoral activity but again the immunity produced does not contributesignificantly to PA's activity at the amount of PA used Likewise, anamount of PA that is substantially independent of Fc binding means thatFc binding does not contribute significantly to PA's activity at theamount of PA used. In other words, removing or impairing the Fc functionof PA would not destroy PA's activity at the amount used. In general, atthe low amounts of PA used, superantigen activity, stimulation ofhumoral immunity or Fc binding of PA does not contribute significantlyto PA's activity.

Superantigen activity is typically characterized by the stimulation ofnon-specific subsets of T cells to proliferate. That is, T cellproliferation is largely independent of epitope specificity.Superantigen activity typically stimulates about 5-10% of T cells toproliferate whereas a conventional antigen may stimulate about 1 in 10⁶cells in an individual. Superantigen activity may therefore be assayedby determining numbers of T cells that are stimulated to proliferate.Examples of such assays are described, for example, in Johnson et al.,Scientific American, April 1992. pp. 92-101; and Kotzin et al., Adv.Immunol. 54:99 (1993). Superantigen and FC binding assays are described,for example, in Romagnani et al., J. Immunol. 129:596 (1982). FC bindingassays are described, for example, in Langone J J, Adv. Immunol. 32:157(1982). Stimulation of humoral immunity assays are described, forexample, in Leonetti et al., J. Exp. Med. 189:1217 (1999).

Methods of the invention can therefore be practiced using thecompositions of the invention. For example, in one embodiment, aneffective amount of PA is a dose of about 0.1 picogram to about 1microgram. In another embodiment, an effective amount of PA is a dose ofabout 1 picogram to about 1 microgram. In yet another embodiment, aneffective amount of PA is a dose of about 10 picograms to about 1microgram. In still another embodiment, an effective amount of PA is adose of about 10 picograms to about 0.1 microgram. In additionalembodiments, an effective amount of PA is a single dose of about 10picograms to about 0.1 microgram.

Compositions may be administered systemically or locally by any route.For example, PA may be administered intravenously, orally (e.g.,ingestion or inhalation), intramuscularly, intraperitoneally,intradermally, subcutaneously, intracavity, intracranial, transdermally(topical), parenterally, e.g. transmucosal and rectally. Compositions ofthe invention including pharmaceutical formulations can be administeredvia a microencapsulated delivery system or packaged into an implants foradministration.

Compositions further include pharmaceutical formulations containing PAin an amount having one or more of the activities disclosed herein. Invarious embodiments, a pharmaceutical formulation includes PA in anamount sufficient to re-regulate or normalize aberrant or undesirablehumoral or cellular immune response (modulating lymphocyteproliferation, apoptosis or differentiation), inhibit, reverse,ameliorate or reduce autoimmunity, inflammation or an inflammatoryresponse, or at least a portion of tissue damage caused by anundesirable or aberrant immune response (inhibiting or preventingdisease progression, promoting or enhancing disease reversal or tissueregeneration), normalize T or B splenocyte numbers or their response toone or more mitogens, without substantial superantigen activity, withoutsubstantial stimulation of humoral immunity or substantially independentof Fc binding, and a pharmaceutically acceptable carrier or excipient.

As used herein, the terms “pharmaceutically acceptable” and“physiologically acceptable” refer to carriers, excipients, diluents andthe like that can be administered to a subject, preferably withoutproducing excessive adverse side-effects (e.g., nausea, abdominal pain,headaches, etc.). Such preparations for administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions.

Pharmaceutical formulations can be made from carriers, diluents,excipients, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with administration to a subject. Such formulations canbe contained in a tablet (coated or uncoated), capsule (hard or soft),microbead, emulsion, powder, granule, crystal, suspension, syrup orelixir. Supplementary active compounds and preservatives, among otheradditives, may also be present, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.

A pharmaceutical formulation can be formulated to be compatible with itsintended route of administration. Thus, pharmaceutical formulationsinclude carriers, diluents, or excipients suitable for administration byroutes including intraperitoneal, intradermal, subcutaneous, oral (e.g.,ingestion or inhalation), intravenous, intracavity, intracranial,transdermal (topical), parenteral, e.g. transmucosal and rectal.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following: a sterile diluentsuch as water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical formulations suitable for injection include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. Fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride can beincluded in the composition. Prolonged absorption of injectableformulations can be achieved by including an agent that delaysabsorption, for example, aluminum monostearate or gelatin.

For oral administration, a composition can be incorporated withexcipients in the form of tablets, troches, or capsules, e.g., gelatincapsules. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included in oral formulations. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or flavoring.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including materials that slowly degradewithin the body and in turn release the active ingredient(s). Forexample, a time delay material such as glyceryl monostearate or glycerylstearate alone, or in combination with a wax, may be employed.

Additional formulations include biodegradable or biocompatible particlesor a polymeric substance such as polyesters, polyamine acids, hydrogel,polyvinyl pyrrolidone, polyanhydrides, polyglycolic acid,ethylene-vinylacetate, methylcellulose, carboxymethylcellulose,protamine sulfate, or lactide/glycolide copolymers,polylactide/glycolide copolymers, or ethylenevinylacetate copolymers inorder to control delivery of an administered composition. Methods forpreparation of such formulations will be apparent to those skilled inthe art. The materials can also be obtained commercially from AlzaCorporation and Nova Pharmaceuticals, Inc., for example.

The rate of release of a composition can be controlled by altering theconcentration or composition of such macromolecules. For example, thecomposition can be entrapped in microcapsules prepared by coacervationtechniques or by interfacial polymerization, for example, by the use ofhydroxymethylcellulose or gelatin-microcapsules orpoly(methylmethacrolate) microcapsules, respectively, or in a colloiddrug delivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Additional pharmaceutical formulations appropriate for administrationare known in the art and are applicable in the methods and compositionsof the invention (see, e.g., Remington's Pharmaceutical Sciences (1990)18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12thed., Merck Publishing Group, Whitehouse, N.J.; and PharmaceuticalPrinciples of Solid Dosage Forms, Technonic Publishing Co., Inc.,Lancaster, Pa., (1993)).

Compositions of the invention can include combinations of othercompositions, and be included in the pharmaceutical compositions of theinvention. For example, a drug that reduces an inflammatory response orinflammation or that stimulates differentiation of a cell can beincluded with a low amount of PA. Exemplary drugs include steroidal(SAI) and non-steroidal anti-inflammatory's (NSAI), for example, acorticosteroid, a cox-2 inhibitor, or drugs that affect the immunesystem such as chemokines and cytokines such as interleukins andinterferons.

Compositions of the invention, including pharmaceutical formulations canbe packaged into kits, which optionally can contain instructions foruse, for example, practicing a method of the invention. The inventiontherefore provides kits. In one embodiment, a kit includes one or morecompositions of the invention (e.g., PA), including pharmaceuticalformulations, packaged into suitable packaging material. In additionalembodiments, a kit includes a label or packaging insert for practicing amethod of the invention. Thus, in one embodiment, a kit includesinstructions for treating a subject having or at risk of having animmune disorder or dysfunction, in vitro, in vivo, or ex vivo. In yetadditional embodiments, a kit includes a label or packaging insertincluding instructions for treating a subject having an autoimmunedisorder with low amounts of PA in vivo, or ex vivo.

As used herein, the term “packaging material” refers to a physicalstructure housing the components of the kit. The packaging material canmaintain the components sterilely, and can be made of material commonlyused for such purposes (e.g., paper, corrugated fiber, glass, plastic,foil, ampules, etc.). The label or packaging insert can includeappropriate written instructions, for example, practicing a method ofthe invention. Kits of the invention therefore can additionally includeinstructions for using the kit components in a method of the invention.

Instructions can include instructions for practicing any of the methodsof the invention described herein. Thus, invention pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration to a subject. Instructions mayadditionally include indications, a satisfactory clinical endpoint, anyadverse symptoms that may occur, or additional information required bythe Food and Drug Administration for use on a human subject.

The instructions may be on “printed matter,” e.g., on paper or cardboardwithin the kit, on a label affixed to the kit or packaging material, orattached to a vial or tube containing a component of the kit.Instructions may comprise voice or video tape which can optionally beincluded on a computer readable medium, such as a disk (floppy disketteor hard disk), optical CD such as CD- or DVD-ROM/RAM, magnetic tape,electrical storage media such as RAM and ROM and hybrids of these suchas magnetic/optical storage media.

Invention kits can also include one or more drugs that provide asynergistic or additive effect or that reduce or ameliorate one or moresymptoms of a drug or disorder. For example, a drug that reduces aninflammatory response or inflammation may be included. Exemplary drugsinclude steroidal (SAI) and non-steroidal anti-inflammatory's (NSAI),for example, a corticosteroid, or a cox-2 inhibitor. Invention kits canadditionally include a buffering agent, a preservative, or a stabilizingagent. The kit can further include control components for assaying anactivity or effect of treatment. Each component of the kit can beenclosed within a separate individual container. For example, a kit caninclude a single unit dosage of a low amount of PA as set forth herein(e.g., from less than 1 μg to 1 pG). Alternatively, a kit can includemultiple unit dosage forms of a low amount of PA. For example, each ofthe multiple unit dosage forms would contain a low amount of PA in aseparate individual container (e.g., each unit dose of PA would be fromless than 1 μg to 1 pG per dose). Kit components can be in a mixture ofone or more containers and all of the various containers can be withinsingle or multiple packages.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All publications, patents and other references cited herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a lymphocyte” includes a plurality of such cells.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

Example 1

This example describes an animal inflammation (arthritis) model andhistological data indicating that PA administered at very lowconcentrations can reduce inflammation and inhibit or reverse tissuedamage caused by inflammation.

Three separate studies with three groups of five animals each (a totalof 15 animals per treatment group). The first group is control, injectedwith phosphate buffered saline) PBS carrier. The second group received100 μg Enbrel per mouse per day. This was the optimal Enbrel dose asdescribed by the manufacturer (Immunex, Corp., Seattle, Wash.). Thethird group was injected with 10 picograms (pG) of PA in PBS carrier onMonday, Wednesday, and Friday during the treatment period(Amersham/Pharmacia Biotech, Piscataway, N.J.). PA may also be obtainedfrom Sigma-Aldrich, St. Louis, Mo.; Pierce Chemical Co., Pittsburgh,Pa.; and Calbiochem, San Diego, Calif.

Table 1 below summarizes the clinical index data for the control groupindicating a progressive inflammatory response in these susceptibleanimals. The response does not peak or plateau within the time limitsused and control animals were sacrificed when the response jeopardizedtheir health status.

TABLE 1 Control Clinical Index Reading - CIA Model Mean S.D. SEM N Day 00 0 0 0 0 0 0 30 1 0.00 0.00 0.00 5.00 2 0.00 0.00 0.00 25.00 3 0.250.50 0.25 4.00 4 0.08 0.28 0.06 25.00 5 1.00 0.94 0.30 10.00 7 0.96 1.060.21 25.00 8 1.60 1.51 0.48 10.00 9 1.40 1.35 0.30 20.00 10 3.52 3.440.69 25.00 15 7.80 5.63 2.52 5.00 16 10.80 3.11 1.39 5.00 18 2.25 1.590.35 20.00 19 2.58 1.50 0.34 19.00 23

Table 1: Raw data of mean clinical index measurements for 15 controlanimals treated by the CIA protocol. This data is pooled data from 3separate studies.

Table 2 shows similar data for the caliper measurements of the paws ofthe control group animals during the same time period. These data mirrorthe clinical index data with the exception that a plateau in theresponse appears after day 10. This response plateau corresponds withthe kinetics of typical antibody induced inflammatory responses.

TABLE 2 Control Paw Measurement - CIA Model Mean S.D. SEM N 165.80 9.312.08 20.00 0 164.95 11.91 1.52 61.00 1 168.50 6.13 1.37 20.00 2 168.508.90 1.41 40.00 3 172.30 4.75 1.06 20.00 4 171.38 7.26 1.15 40.00 5167.00 16.12 2.55 40.00 7 182.35 19.06 3.01 40.00 8 176.44 19.56 3.0541.00 9 207.45 36.93 8.26 20.00 10 212.28 38.03 6.01 40.00 15 204.3031.25 6.99 20.00 16 212.95 36.18 8.09 20.00 18 217.40 44.90 10.04 20.0019 224.30 44.01 9.84 20.00 23

Table 2: Raw data of mean caliper paw measurements for 15 controlanimals treated by the CIA protocol. This data is pooled data from 3separate studies.

Histological analysis of knee and ankle of a control animal taken on day15 of the inflammatory response indicated extensive immune infiltrationand tissue destruction. Immunocytes accumulated in the synovium ofcontrol animal. Enbrel treated animals at day 35 showed continued immuneinfiltration in the synovium of knee and ankle and evidence of continuedtissue destruction.

Table 3 summarizes the histological examination results of the knee andankle joints of control, untreated DBA/1 animals. These ratings areassigned on a blinded basis by the histologist on a continuous scalefrom 1 to 10 with 1 representing a “normal” histological appearance and10 a high degree of damage. Knee and ankle joints of the control animalsafter collagen induction show maximal tissue damage (“10”s). Thiscorrelates with both the Clinical index (Table 1) and physicalmeasurement data (Table 2).

TABLE 3 Histology Rating of CIA Model - Control Animals Histology TissueRating N Ankle 10 15 Knee 10 15

PA treated animals at day 35 of the inflammatory response showed muchless evidence of immune infiltration and only slight evidence of tissuedestruction. Only a few host immunocytes were present in the synoviumand there was no evidence of tissue fragments or tissue destruction.These data therefore demonstrate that PA reduces acute inflammation inthe CIA model. These data also demonstrate that PA reduces tissue damageor promotes tissue repair more than Enbrel.

Tables 4 and 5 show the results of 3 separate studies testing the effectof PA (10 pG/injection, M/W/F). Enbrel was tested for comparison; theresults obtained were comparable with published results. PA treatmentwas very effective, reaching significance at approximately 10% of thenumber of animals required for the Enbrel standard. The results for thetotal clinical index (Table 4) and the physical measurements (Table 5)are comparable.

TABLE 4 PA Treatment on the CIA model (Total Clinical Index) PooledData: C.I. Treatment Day Mean N P[1 − tail] Control 15 3.52 25 17 3.8825 19 2.25 20 Protein-A 15 1.92 25 0.03 17 1.95 24 0.02 19 1.40 20 0.04Enbrel 15 2.79 24 0.17 17 3.41 24 0.30 19 2.85 20 0.11

TABLE 5 PA Treatment on the CIA model (Measurement) Pooled Dat: MeasTreatment Day Mean N P[1 − tail] Control 15 212.3 40 17 218.1 40 19217.4 20 Protein-A 15 195.4 36 0.02 17 191.4 36  0.001 19 197.2 20 0.05Enbrel 15 204.2 36 0.18 17 209.1 36 0.18 19 230.3 20 0.19

Table 6 shows the results of a histological assessment of DBA/1 micesacrificed at 1, 2 and 3 weeks during their treatment regimen. Both PAand Enbrel treatments show significant damage at the tissue level. Thesedata demonstrate that in spite of the significant decreases in Totalclinical index and paw measurements (Tables 4 and 5) there is stilldamage at the tissue level. PA treatment appears to delay tissue damage(treatment week one versus control) but does not prevent the damage.

TABLE 6 PA treatment and histological damage Control PA Enbrel 1 wkAnkle: 10 Ankle: 8 Ankle: 1-5 Knee: 10 Knee: 0-5 Knee: 0-10 2 wk Ankle:10 Ankle: 10 Ankle: 10 Knee: 10 Knee: 10 Knee: 10 3 wk Ankle: 10 Ankle:10 Ankle: 10 Knee: 10 Knee: 10 Knee: 10

DBA/1 mice induced with Type II Collagen as per the CIA model protocolwere treated immediately after the second antigen injection-solventcarrier (PBS) for the control, PA (at 10 pG per injection M/W/F), andEnbrel (100 ug/injection every day).

Table 7 shows histological results after extending treatment to 35 days.Control mice were sacrificed at 18 days for humanitarian reasons andtheir 18 day data are included for comparison. Enbrel treatment showedno amelioration of the histological damage. In contrast, PA treatmentreversed histological damage at 14-21 days. This histological datacorrelates with the Total Clinical Index of these animals. Thus, PAtreatment significantly reduced the severity of the acute inflammatoryresponse and continuing treatment reversed the existing tissue damagecaused by the response.

TABLE 7 PA extended Treatment and Histological assessment Control @ PA @35 Enbrel 18 Days Days @35 Days rating Ankle: 10 Ankle: 0-1 Ankle: 10Knee: 10 Knee: 0-1 Knee: 10

The histological assessment of these tissues included low, medium, andhigh magnification assessment. In both control (at 18 days, time ofsacrifice) and Enbrel treatment groups the synovium had large numbers ofactivated lymphocytes, whereas the PA group at day 35 of treatment hadfew small lymphoid cells which were similar in number and morphology tothose in the DBA/1 animals prior to Type II collagen antigen induction.

In sum, these data demonstrate that paw measurements and the clinicalindex assessments document the induced inflammatory response in the CIAanimal model; that PA reduces the inflammatory response during the acutephase (P values vs control <0.05) and reverses the histological damagecaused by the response by day 35 of treatment; that PA has itsameliorative effect at concentrations and dosing schedules predicted bythe BXSB animal model and Tissue Culture assessments (discussed furtherbelow); and that Enbrel does reduce inflammation on Day 14 (notsignificantly with N=15) but does not reduce inflammatory damageobserved at day 35, indicating that PA is more effective than Enbrel.

Example 2

This example describes data indicating that the mechanism of action(MOA) of PA appears distinct from Enbrel.

The accepted MOA for the CIA animal model is competitive inhibition ofthe expression of α-TNF. Enbrel is a known α-TNF inhibitor. Regressionanalysis of the Enbrel data indicated a delay in the onset of theinflammatory response. In contrast, regression analysis of the PA datasuggested an alteration in the inflammatory process itself. The MOA ofPA therefore does not appear to be primarily through α-TNF inhibition.In addition, PA is not only more effective than Enbrel in reducing theinduced inflammatory response in treated animals but is also capable ofreversing pre-existing tissue damage caused by that response.

While not being bound by any theory, PA may therefore “modulate” a basalcontrol mechanism responsible for integrating immune-dependentresponses. This MOA would encompass α-TNF inhibition but from aself-regulatory perspective instead of simple target moleculecompetitive inhibition. Such a MOA is predicted to have the followingproperties:

1) small amounts required—a regulatory effect on a primitive controlmechanism that “branches” to influence additional mechanisms;

2) self-regulatory—if PA acts at an early control point then the systemwill have the ability to regulate the intensity and direction of themechanism resulting in few if any side effects;

3) pleiotropic target i.e. non-cell-lineage specific—if PA control pointis early then the subsequent control should be diverse, and theconcentration of PA and dosing schedule should be constant; and

4) PA effector molecule structure should be found in association with anumber of more complex structures.

Example 3

This example describes data indicating that PA has multiple modulatoryactivities in an animal model characterized by a combined autoimmunedeficiency disease having a genetic basis resulting in early death. Inparticular, PA prevents early onset of wasting, expansion of the spleniccompartment, regulates humoral immunity (autoantibodies), cellularimmunity (TH₁/TH₂ balance) and lymphoid cell differentiation as well asameliorating tissue damage caused by the disease processes.

The BXSB murine model is a gene (Yaa) based animal model that manifestswith early death in males, typically from kidney failure. This model isconsidered in the literature as an analog for human systemic lupus. Thegene defect expresses as a series of inter-related progressive systemicautoimmune diseases having the following pattern: thymic atrophy,anti-nuclear antibody, liver disease, arthritic disease, kidney diseaseand early death.

Because this is a genetic model with multiple outcomes previous studiesby other investigators have concentrated on single aspects of thedisease. The effect of PA on multiple aspects of the disease processstudied herein include:

-   -   1. overall effect on the animal's physiology        -   a. growth curves        -   b. histology of thymus, liver, brain, kidney, ankle, and            knee    -   2. immune regulation—cellular proliferation/apoptosis        -   a. splenic size, and        -   b. cell count    -   3. lymphocyte dynamics        -   a. T/B responses to mitogenic stimuli    -   4. lymphocyte function        -   a. Humoral immunity            -   i. Ig-PFC production            -   ii. Auto-antibody: ANA, anticardiolipin        -   b. Cellular Immunity            -   i. Natural killer        -   c. Cell surface markers        -   d. Cellular cytokines

Study design:

-   -   1. Chronic Treatment, Abnormal: groups of 25 male BXSB        (control+4 treatment groups) were treated with PA on M/W/F over        a 15 week period with periodic peel-off sacrifices (usually        every 3 weeks of treatment)    -   2. Chronic Treatment, Normal: groups of 15 male C57BL/6J were        treated with PA on M/W/F over a 15 week period with periodic        peel-off sacrifices (usually every 3 weeks of treatment)    -   3. Acute Treatment, Abnormal: groups of 15 male BXSB were        treated for 3 weeks with amounts of PA determined above, and        then animals were sacrificed 3, 6 and 9 weeks post treatment.    -   4. Acute Treatment, Normal: groups of 15 male C57BL/6J were        treated for 3 weeks with amounts of PA determined above, and        then animals were sacrificed 3, 6 and 9 weeks post treatment.

Determination of PA Concentration:

PA amounts were administered to BXSB over eight logs of concentration,from 1 μG/injection to 10⁻⁷ μG/injection. The results indicate two PAoptima, one at 0.01 μG/injection and another at 10⁻⁵ μG/injection. Theshape of the dose-response curves is Gaussian. For the sake of claritythe data presented is for 10⁻⁵ μG/injection (FIG. 1).

FIG. 1 shows the weight gain of BXSB males following PA administration.The weights were taken each time the animals were injected with eithercarrier or PA. Panel A is the weight gain growth curve for a normalC57BL/6J mouse strain, and is presented to show the typical shape of anormal weight gain growth curve. Panel B shows the cumulative sum ofweight gain data from 25 control BXSB male mice. This curve shows aweight peak at approximately 4 months of age followed by a decrease inbody weight, which corresponds to the reported onset of the BSXBautoimmune disease. The decrease in body weight leads to the “wasting”syndrome linked to immune complex deposition in the kidney.

Panel C shows the effect of chronic administration of PA at the optimaltwo concentrations (8 concentrations studied). Both the 0.01μG/injection and 0.00001 μG/injection show significant changes in boththe shape of the weight gain growth curve comparing better with thenormal logistic shape than with the BXSB control and the average weightof both the treated animal groups is significantly higher than control(X=24.16 P=0.0002, for the 10⁻⁵ μG dose).

In order to analyze these growth curves and compare them when treatinggroups of BXSB animals with PA a regression analysis was performed onthe data. The C57BL/6J growth data presented above, is best representedby the following cubic equation:

C57BL/6J Control—y=−0.0286x ³+0.4067x ²−0.8452x+16.816 [R ²=0.9886]

The R-squared value indicates an extremely good fit between the actualdata and the line equation. The equation itself is a relatively simpleextraction of a “logistic” shaped curve, which is typical of normalgrowth curves (FIG. 1).

There are four separate data sets for the control BXSB growth curve. Alldata sets agree internally and the overall equation for the pooled datais as follows:

Pooled BXSB—y=−0.0001x ³+0.0034x ²+0.1851x+18.746 [R ²=0.9384]

This equation is a quantitative representation of the differences incurve shape noted between FIG. 1, panels A and B. The cubic andquadratic function define the overall shape and the very low valuesdefine the rate of increase in weight and the “wasting” phase where theweights are seen to decrease as a function of time. In this study theaim is not to make the BXSB growth curve “look like” the C57BL/6J curve,as each strain has there own specific growth characteristics, but ratherto increase the rate of growth initially and decrease or eliminate the“wasting phase” evident in FIG. 1, panel D.

Table 8 shows the fitted equations for the various PA treatment groupsof BXSB animals. The slope of the initial growth curve, which indicatesgrowth rate, is severely restricted in BXSB control animals. Theinflexion point in BXSB control animals indicates the lethal effect ofthe disease process. The results indicate that PA treatment has apositive effect on both growth rate and lethality in a dose dependentmanner.

TABLE 8 PA effect on the growth kinetics of BXSB Treatment Growthequation Pooled BXSB y = −0.0001x3 + 0.0034x2 + 0.1851x + 18.746 R2 =0.9384 Rx 1.0 PA y = 4E−06x3 − 0.4942x2 + 18171x − 2E+08 R2 = 0.9087 Rx0.1 PA y = −1E−04x3 + 10.697x2 − 392306x + 5E+09 R2 = 0.9463 Rx 0.01 PAy = 3E−05x3 − 2.8056x2 + 102980x − 1E+09 R2 = 0.918 Rx 0.001 PA y =1E−05x3 − 1.3946x2 + 51161x − 6E+08 R2 = 0.9773 Rx 0.0001 PA y =−4E−05x3 + 4.2053x2 − 154454x + 2E+09 R2 = 0.9487 Rx 0.00001 y =−1E−05x3 + 1.4883x2 − 54653x + 7E+08 PA R2 = 0.8665 Rx 0.000001 y =3E−05x3 − 2.9251x2 + 107455x − 1E+09 PA R2 = 0.8682 Rx 0.0000001 y =−5E−06x3 + 0.5628x2 − 20666x + 3E+08 PA R2 = 0.9474

Histological Analysis:

The histological deterioration of various organs of the BXSB mouse modelis the hallmark of a combined autoimmune deficiency disease. Thissyndrome is multi-facetted and involves a number of organs. The organdamage is progressive and individual damage may vary according to tissuetype.

Table 9 shows the results from the histological analysis of higheramounts of PA (1 to 0.001 μg/injection) and indicates that PA changedthe onset and/or severity of the histological changes in a dosedependent manner. Table 10 shows similar data for lower amounts of PA(0.00001 to 0.0000001 μg/injection). Both PA doses exhibited thegreatest improvement in organ histology.

TABLES 9 and 10 Histology of BXSB BXSB Rx 0.01 μg Rx 0.001 μg TissueControl Rx 1 μg PA Rx 0.1 μg PA PA PA Thymus 4+ 4+ 4+ 4+ 4+ (10-20 wks)(16-20 wks) (10-20 wks) (16-20 wks) (12-13 wks) Liver local N/A locallocal local (double nuclei) inflammation inflammation inflammationinflammation 20 wks 19 wks 10 wks 20 wks Kidney normal normal normalnormal normal Knee normal normal normal inflammation normal 16 wks Anklefibers inflammation inflammation inflammation normal disintegrating 19wks 10 wks 16 wks 20 wks Rx 0.0001 μg Rx 0.00001 μg Rx 0.000001 μg Rx0.0000001 μg Tissue BXSB Control PA PA PA PA Thymus 4+ normal 4+ 4+ 4+(11-22 wks) (11-22 wks) (22 wks) (22 wks) Liver local normal localnormal local (double inflammation inflammation inflammation nuclei) 20wks 19 wks 11 wks Kidney normal normal normal normal normal Knee normalnormal normal normal normal Ankle normal normal normal normal normal

The results in Tables 9 and 10 indicate that PA treatment of BXSB micewas able to decrease autoimmune damage in a dose dependent fashion.Treatment delayed the onset of thymic atrophy, and reduced the severityof liver and joint inflammation. The kidneys remained with in normalrange. The tissues of PA treated animals exhibited a reversal of thedamage present in BXSB control tissues, although they were notcompletely normal as compared to C57BL/6J animals. The optimal PA dosewas 0.0001 μg, which is consistent with other assay systems (e.g.,Ig-PFC production, NK activity, mitogen response pattern, cytokineproduction, autoantibody production, spleen size, histologicalimprovements).

In sum, the studies indicate that PA prevents the early onset ofwasting, and ameliorates or reverses damage caused by autoimmune diseaseprocesses in tissues including thymus, liver, kidney, knee and ankle.

Example 4

This example describes data indicating that PA has immune modulatoryactivity (e.g., proliferation, apoptosis or differentiation) in spleenreflected by inhibition of spleenic expansion and splenocyte cellnumbers.

Spleens were removed from animals during the studies. Normal C57BL/6Jspleen was a reference point. Spleens from untreated BXSB animals weresignificantly enlarged. In contrast, PA treated BXSB animal spleensresembled the size of normal C57BL/6J spleen.

The enlarged spleens in untreated BXSB animals is known assplenomegalia, and is the result of either a massive systemic infectionprocess or of aberrant cellular apoptosis or proliferation. Becausethese animals are maintained in a sterile environment the enlargedspleen is likely the result of faulty cell growth/death control. Anotherexplanation for enlarged spleen would be that the cells had increased intheir individual size, a process known as blastosis. Examination of thefluorescently activated cell sorting (FACS) data to measure cell sizeruled out this possibility. FACS analysis revealed that although a trendto slightly larger cells was observed in the BXSB controls, nostatistically significant increase in cell size was observed. Consistentwith these findings is that BXSB splenocyte numbers are more thanfive-times those of normal animals (Table 11, top).

TABLE 11 PA Effect on Splenomegalia BXSB control C57BL/6J DBA/2J MEAN5.E+08 9.E+07 1.E+08 SEM 4.E+07 7.E+06 6.E+06 N 20 20 20 Rx 1.0 PAP-value Rx 0.1 PA P-value Rx 0.01 PA P-value Rx 0.001 PA P-value MEAN1.E+08 8.68E−03 1.E+08 7.32E−04 7.E+07 1.02E−06 1.E+08 8.92E−06 SEM7.E+07 5.E+07 4.E+07 4.E+07 N 3 3 5 4 Rx E−04 PA P-value Rx E−05 PAP-value Rx E−06 PA P-value Rx E−07 PA P-value MEAN 3.E+08 1.73E−021.E+08 6.70E−05 3.E+08 9.54E−02 2.E+08 7.26E−05 SEM 6.E+07 3.E+07 1.E+084.E+07 N 4 3 4 5

Table 11 (top) shows the contents of the spleens of both control mousestrains, C57BL/6J and DBA/2J, and BXSB males. BXSB splenocyte cellnumbers are more than 5 times those found for either of the normalstrains. PA treatment significantly reduced BXSB splenocyte numbers(Table 11, bottom).

In sum, the studies indicate that PA regulates expansion of the spleeniccompartment (proliferation/apoptosis) caused by the autoimmune diseaseand splenocyte numbers. PA treatment both reduced spleen size andsplenocyte numbers significantly in a dose dependent fashion.

Example 5

This example describes data indicating that BXSB animals exhibitaberrant splenocyte differentiation or proliferation or apoptosis. Thisdata therefore indicates that PA activity in BXSB spleen includesre-regulating aberrant/deficient splenocyte differentiation orsplenocyte proliferation/apoptosis (i.e. restoring normal cellproliferation or apoptosis).

Mitogens are lectins that have the non-specific capacity to stimulatecellular division in general populations of cells (e.g., T and/or Blymphocytes). Lectins are found in plants and animals and are bestcharacterized as precursors to modern day antibody molecules. Lectinsare used for intracellular and other forms of communication.

To determine whether the T-lymphocyte compartment in BXSB mice isaberrant, two T-cell specific and two B-cell specific mitogens were usedto study the response of splenocytes isolated from BXSB mice. 7different mitogen concentrations and 4 kinetic time points were used totest BXSB animals' splenocyte response in terms of the responseamplitude (an indication of the number of cells present), and secondly,the optimal stimulation concentration (an indication of thedifferentiation status of the cells).

In brief, BXSB and control mice were sacrificed periodically during thetreatment regimen and their spleens removed. Splenocyte cell suspensionsat 1 to 2×10⁶/ml are dispensed into 96 well plates. Mitogens (10 μG/10uL to 0.01 μG/10 uL) were added in triplicate to the wells and thecultures harvested at 24 hour intervals from 24 to 96 hours of culture.All cultures were treated with tritiated thymidine for 16 hours prior toharvest. The DNA, including the newly synthesized radiolabeled DNA, isextracted on glass fiber filters and the radioactivity determined byliquid scintillation counting.

TABLE 12 Normal (C57BL/6) mitogen responses (Stimulation Index, S.I.)10.00 5.00 1.00 0.50 0.10 0.05 0.01 PHA: SI MEAN 24.56 29.54 33.39 19.953.07 1.79 1.03 S.D. 11.39 13.47 14.00 11.47 3.40 1.69 0.57 SEM 2.15 2.552.65 2.17 0.64 0.32 0.11 Count 27 27 27 27 27 27 27 Con-A: SI MEAN 0.571.69 48.07 11.79 1.83 2.00 2.39 S.D. 0.27 1.50 32.63 15.80 1.94 1.082.99 SEM 0.05 0.28 6.17 2.99 0.37 0.20 0.56 Count 27 27 27 27 27 27 27SEB: SI MEAN 18.70 15.79 10.61 7.87 5.23 3.41 1.47 S.D. 11.49 8.69 6.145.57 4.57 2.47 0.55 SEM 2.17 1.64 1.16 1.05 0.86 0.47 0.10 Count 27 2727 27 27 27 27 LPS: SI MEAN 105.66 103.65 94.60 85.92 69.95 59.05 39.44S.D. 46.55 46.89 46.30 52.67 41.29 36.17 29.92 SEM 8.80 8.86 8.75 9.957.80 6.84 5.66 Count 27.00 27.00 27.00 27.00 27.00 27.00 27.00

Table 12 shows the mitogen responses of C57BL/6J mice tophytohemaglutinin (PHA), concanavalin A (Con-A), staphylococalenterotoxin B (SEB), and lipopolysaccharide (LPS). PHA and Con-Astimulate basic T lymphocyte populations to proliferate in a dosedependent fashion, whereas SEB, and LPS stimulate the dose dependentproliferation of B-lymphocyte populations.

TABLE 13 BXSB mitogen responses 10.00 5.00 1.00 0.50 0.10 0.05 0.01 PHA:SI Mean 4.63 2.17 3.64 4.29 3.77 2.55 1.95 S.D. 6.29 1.40 1.56 2.11 1.760.81 0.88 SEM 0.87 0.19 0.22 0.29 0.24 0.11 0.12 N 39 39 39 39 39 39 39Con-A: SI Mean 0.99 2.42 6.34 7.08 3.93 1.88 1.67 S.D. 1.02 2.07 4.323.69 3.46 0.97 0.93 SEM 0.14 0.29 0.60 0.51 0.48 0.14 0.13 N 39 39 39 3939 39 39 SEB: SI Mean 6.05 6.10 5.21 4.70 3.00 2.53 1.91 S.D. 3.77 3.182.69 3.48 1.66 1.85 1.04 SEM 0.52 0.44 0.37 0.48 0.23 0.26 0.14 n 39 3939 39 39 39 39 LPS: SI Mean 26.40 27.76 29.58 24.83 10.85 7.08 3.97 S.D.18.99 20.58 20.78 14.66 5.50 3.76 1.64 SEM 2.63 2.85 2.88 2.03 0.76 0.520.23 N 39 39 39 39 39 39 39

Table 13 shows the results from the mitogen study of BXSB control.Statistical analyses indicates that all mitogen responses aresignificantly lower than C57BL/6J control. In addition, the shape of thePHA and Con-A response curves for BXSB control are different from normalC57BL/6J control. For both there was a shift to lower mitogenconcentrations to achieve a peak response. These changes in responsecurve shape have been associated with changes in differentiation status.

TABLE 14 Statistical Comparison of BXSB Mitogen Responses to Control10.00 5.00 1.00 0.50 0.10 0.05 0.01 PHA 1.E−10 2.E−11 2.E−11 5.E−075.E−02 N.S. N.S. Con-A N.S. N.S. 6.E−07 6.E−02 N.S. N.S. N.S. SEB 2.E−054.E−06 4.E−05 2.E−04 6.E−04 1.E−03 N.S. LPS 5.E−09 2.E−08 5.E−07 5.E−068.E−08 8.E−08 N.S.

Table 14 shows a statistical comparison of control BXSB. Mitogenresponses to control C57BL/6. In almost every case, the amplitude of themitogen response in BXSB is suppressed indicating a dimunition of themature cell population and a shift in the optimal response concentrationto a lower value. These data therefore indicate aberrant differentiationin control BXSB splenocytes which leads to over-proliferation ordecreased apoptosis of splenocytes.

TABLE 15 PHA Response as a Function of PA treatment PHA: S.I. 10.00 5.001.00 0.50 0.10 0.05 0.01 1 PA 2.23 3.27 8.64 11.52 10.91 4.63 1.86 0.1PA 1.08 1.82 4.13 5.25 4.76 2.95 2.62 0.001 PA 1.52 1.21 2.70 4.41 3.902.57 1.94 0.0001 PA 5.53 4.03 9.65 15.41 10.91 3.91 1.95 1E−04 PA 5.712.98 5.14 6.82 6.22 2.73 1.66 1E−05 PA 7.73 7.00 8.34 9.80 7.26 3.471.89 1E−06 PA 2.70 3.13 4.47 4.82 3.84 3.29 2.20 1E−07 PA 5.08 5.84 3.414.22 3.90 2.37 2.00

TABLE 16 Con-A Response as a Function of PA treatment Con-A: S.I. 10.005.00 1.00 0.50 0.10 0.05 0.01 1 PA 1.42 4.88 23.61 36.83 6.57 2.70 2.070.1 PA 1.29 5.43 8.51 8.06 2.00 2.20 1.64 0.001 PA 1.34 3.31 8.26 17.49.14 3.33 1.92 0.0001 PA 1.92 2.05 27.27 51.59 10.40 2.10 2.02 1E−04 PA0.56 1.31 7.29 15.26 6.87 4.56 1.79 1E−05 PA 0.76 3.10 5.31 14.29 7.692.25 1.34 1E−06 PA 1.01 2.34 5.04 15.98 8.03 2.85 2.52 1E−07 PA 1.652.90 9.05 22.83 8.10 2.17 2.05

TABLE 17 SEB Response as a Function of PA treatment SEB: S.I. 10.00 5.001.00 0.50 0.10 0.05 0.01 1 PA 13.39 11.88 12.41 8.39 6.42 4.73 3.76 0.1PA 6.25 6.57 4.29 4.15 2.37 1.68 1.32 0.001 PA 7.51 7.54 10.06 7.37 5.765.28 3.82 0.0001 PA 14.60 12.60 9.55 5.72 4.66 4.21 2.05 1E−04 PA 8.015.15 6.48 4.55 5.12 2.97 1.34 1E−05 PA 6.92 8.02 7.94 5.28 4.13 3.543.40 1E−06 PA 9.34 8.01 9.74 5.82 4.33 5.73 4.20 1E−07 PA 5.92 5.67 5.886.90 2.33 2.35 2.33

TABLE 18 LPS Response as a Function of PA treatment LPS: S.I. 10.00 5.001.00 0.50 0.10 0.05 0.01 1 PA 52.36 52.74 56.61 49.82 22.00 18.14 8.470.1 PA 20.73 20.47 20.23 16.29 7.47 6.42 3.45 0.001 PA 13.85 15.86 16.5712.71 8.52 6.46 4.76 0.0001 PA 37.85 42.85 41.46 37.76 19.10 15.78 6.411E−04 PA 23.10 37.92 32.05 27.29 13.38 20.52 7.49 1E−05 PA 27.43 27.1827.90 28.69 17.44 16.69 8.88 1E−06 PA 22.67 21.88 17.14 17.60 12.40 9.276.30 1E−07 PA 14.49 16.56 17.95 18.56 10.99 7.20 3.40

Tables 15 to 18 show the effects of PA on the mitogen responses of BXSBsplenocytes. Although the kinetics are complex several conclusions canbe made. That PA (0.5 μG) elevates PHA and Con-A responses indicatesnormalization of T and B cell population dynamics. That PA also restoresmuch of the amplitude of the mitogen responses and the shape of theresponse curves indicates a partial or complete restoration of T celland B cell response to mitogens.

In sum, the studies indicate that the aberrant BXSB splenic lymphocytenumbers as well as typical T and B mitogen responses can be corrected,at least in part, with PA treatment.

Example 6

This example describes data indicating that PA reduces the amount ofautoantibodies likely responsible for tissue destruction in BXSBanimals.

Humoral immunity is responsible for antibody production and issignificant as rheumatoid antibody are found in a significant percentageof RA patients. Abnormal antibodies have been found in the synovium ofsome patients. In the BXSB model, abnormal antibodies are produced.

Spleen cells obtained from control and PA treated BXSB animals werestudied in a plaque forming cell (PFC) assay. In this assay the targetred blood cells are labeled with protein-A which will bind any secretedimmunoglobulin regardless of antigenic specificity thus providing abroad view of humoral immunity.

TABLE 19 BXSB Non-specific Antibody Producing Cells BXSB C57BL/6J Mean60333 611.80 S.D. 33915 658.67 N 45 75.00 SEM 5056 120.26

Table 19 shows the Ig-PFC responses of control BXSB splenocytes, whichis 100× greater than control C57BL/6 splenocytes indicating aberrantantibody production in the BXSB animals.

TABLE 20 PA Treatment and Ig-PFC Production in BXSB Animals BXSB RX 0.1Control Rx 1 PA PA Rx 0.01 PA Rx 0.001 PA Mean 60333 20741 35808 2790930533 N 45 11 12 11 15 S.E.M. 5056 6603 12010 2959 9581 P-values4.22E−05 0.04 5.16E−07 0.01 Rx 0.0001 Rx 0.000001 PA Rx 0.00001 PA PA Rx0.0000001 PA Mean 3850 12310 16278 11042 N 5 10 9 12 S.E.M. 696 20404068 1751 P-values 7.33E−15 2.92E−12 3.11E−08 8.16E−13

Table 20 shows the dose related effect of PA on Ig-PFC production. AllPA amounts significantly reduced the over-production of Ig-PFCs with0.0001 μG PA showing the greatest reduction.

BXSB control splenocytes make and secrete antibodies at enormous levels(61400+−6435 PFC/1E06). This is approximately 100 times the valuesobserved in C57BL/6J normal controls. PA treatment significantly reducesauto-antibody levels in a dose and time dependent manner.

TABLE 21 PA and the Effect on Circulating Auto-antibodies in BXSBAnimals Control Rx 0.01 PA  2 weeks 0.262 0.016 0.318 0.000  5 weeks0.272 0.029 0.360 −0.032 11 weeks 1.317 0.103 1.449 0.125 14 weeks 0.4020.283 0.357 0.206

Table 21 shows the development of auto-antibodies in BXSB animals (thehigher numbers indicate greater amounts of circulating autoantibodies).PA treatment reduces autoantibody levels at all time points measured.

TABLE 22 The Effect of PA Concentration on Auto-antibody Production(ANA) Control Rx 1 PA Rx 0.1 PA Rx 0.01 PA Rx 0.001 PA Mean 0.475 0.3060.176 0.091 0.341 N 11 7 7 9 11 S.E.M 0.14 0.03 0.09 0.03 0.10 P-value0.16 0.07 0.02 0.25 Rx 0.0001 Rx 0.000001 PA Rx 0.00001 PA PA Rx0.0000001 PA Mean 0.229 0.090 0.176 0.098 N 9 12 12 15 SEM 0.04 0.060.03 0.03 P-value 0.02 0.02 0.08 0.02

Table 22 shows that PA-mediated reduction of autoantibodies is dosedependent. The greatest reduction occurs at 0.00001 μG.

In sum, PA regulates the number of non-specific antibodies, reducing theamount of damaging autoantibodies. That PA also reduces antibodyproducing cells in BXSB spleens correlates with this data. Thus, PAtreatment restores, at least in part, humoral immunity.

Example 7

This example describes data indicating that PA reduces cytotoxicityresponse of BXSB mice, re-regulating this response to at or nearbase-line levels.

The cellular component of the immune system is the primary integrator offunction for the entire immune system, supplying the T-cells and B-cellsin their various differentiated forms for both recognition and effectorfunction. In the BXSB animal model there have been reports that the CellMediated Immune (CMI) system is intact. However, contrary to thesereports the data described below indicate that CMI is affected in theBXSB mouse.

BXSB mice, both control and treated with PA, were studied for theirability to recognize and lyse non-specific targets labeled withradio-labeled chromium. In brief, about 200 μl spleen cells (1×10⁷/ml)from control and treated BXSB mice were plated on a microtiter plate inRPMI medium and five two fold serial dilutions were made in RPMI media.P815 cells were radiolabeled with chromium (Cr⁵¹) and added to each wellof a 96 well plate, centrifuged for 12 minutes and then incubated for3-4 hours at 37° C. Cells were re-centrifuged and a 110 μl samplecounted for Cr⁵¹. A more detailed protocol is contained in CurrentProtocols in Immunology, 3.11, Assays for T cell Function.

TABLE 23 Cytotoxicity Response of BXSB and Normal Mice BXSB BXSB Rx 3Control C57BL/6J weeks (acute) E/F % Specific % Specific % SpecificRatio Lysis S.D. Lysis S.D. Lysis S.D.  100:1 122.0 5.0 2.0 0.1 5.43 4.1  50:1 95.1 9.4 −1.2 0.5 4.94 4.4   25:1 93.8 10.3 −1.5 0.6 3.94 3.312.5:1 85.4 8.3 −1.3 0.5 3.04 3.1 6.25:1 50.5 35.1 −1.2 0.2 2.84 3.03.13:1 5.3 5.3 −2.5 0.3

Table 23 shows that normal mice (middle column) have a typical base linelevel of natural killer activity to the P-815 target labeled with Cr⁵¹;the level is between 0 and 3% cytotoxicity at an effector target ratioof 100:1. The left column shows the cytotoxicity response of BXSB miceto the same target; the level of cytotoxicity is extremely high at over50% at an effector/target ratio of 6:1. PA treatment of BXSB over a 3 to15 week period (right column) reduced cytotoxicity to base line levels,i.e. 0-3% at E/T ratio of 100:1 without any regression analysispossible.

In sum, the studies indicate that PA treatment reduces the un-regulatedBXSB cytotoxicity responses by a factor of 20, to at or near controlbase-line levels. Thus, PA treatment restores, at least in part,cellular immunity.

Example 8

This example describes data indicating that PA regulates expression ofCD markers reflecting the regulation of cell differentiation,proliferation or apoptosis of cells of lymphoid lineage.

The expression of clustered determinants (CD markers) indicate thedifferentiation status of lymphoid cells. PA specifically regulatesthese markers. This regulation has direct correlations with the datadescribed above.

TABLE 24A CD Marker Profiles 69+4− 69+4+ 69−4+ 4+8− 4+8+ 4−8+ 69+8−69+8+ 69−8+ BL6 7.6 +− 1.4  7.6 +− 1.6  85 +− 2.9 74 +− 4.4 7.6 +− 1.718 +− 3.2 30 +− 2.3 5.0 +− 0.9 66 +− 2.7 control N 23 23 23 23 23 23 2323 23 BXSB 20 +− 4.1 13 +− 1.5 68 +− 5.1 42 +− 7.7 8.5 +− 1.3 49 +− 6.922 +− 5.7 7.8 +− 1.5 71 +− 6.9 2-10 P 0.009 0.017 0.006 0.002 0.340.0008 0.11 0.07 0.25 one-tail BXSB 44 +− 1.0 20 +− 1.3 36 +− 2.1 16 +−1.3  14 +− 0.8 70 +− 1.1 23 +− 2.3  14 +− 0.7 64 +− 2.2 11-15 P 2.5E−124.8E−5 1.8E−9 1.7E−12 0.002 3.3E−14 0.03 1.7E−6 0.3 one-tail

Table 24A shows data of a number of T-cell CD markers. Approximately 80%of normal splenocytes (BL6 control) are non-activated (CD69−) T-cells(CD69−, CD4+). This population decreases in both young (2-10experimental weeks, and 10 to 18 weeks chronological age) and old (11-15experimental weeks, and 19 to 23 weeks chronological age) BXSB animals.There is also a kinetic effect: the 11-15 week BXSB animals are muchmore severely compromised in terms of their T-cell markers than youngerBXSB animals. This correlates to decreased cellular function andincreased overall death rate.

TABLE 24B CD Marker Profiles cont'd 19+45− 19+45+ 19−45+ 80+25− 80+25+80−25+ C57BL/6J 3 +− 0.6 80 +− 2  18 +− 3  70 +− 3 13 +− 1 18 +− 1 BXSB8 +− 4   60 +− 12 35 +− 11 65 +− 7 17 +− 2 18 +− 5 2-10 wks P N.S. N.S.N.S. N.S. N.S. N.S. [one-tail] BXSB 14 +− 7  64 +− 22 22 +− 15 61 +− 618 +− 2 21 +− 8 11-15 wks P N.S. N.S. N.S. N.S. N.S. N.S. [one-tail]

Table 24B shows data of a number of B-cell CD markers. The first set ofthree markers are indicative of resting B-cells (i.e. non-activated) andthe last three represent activated B cells. These data show that theB-cell population is refractory to both the strain and the stage of thedisease process in the BXSB mice. Thus, it appears that the primarycellular immune component of BXSB combined immunodeficiency diseaseprocess involves T-cells.

FIG. 2 shows data from the effect of chronic treatment (from 3 to 15weeks) of BXSB mice with varying concentrations of PA (1E-03 to 1E-07 μGof PA/injection). The first significant feature of this data is thattreatment of BXSB mice with PA regulates the population of CD69−/CD4+T-cells. The regulation is inter-related with the regulation of theother T-cell markers which is to be expected because one cell populationacts on others in the series (and other cell series) in both feedbackand feed forward mechanisms. There is also a PA dose response effect oncell populations, consistent with the response described above in thefunctional assays; there is a dose-time kinetic response as well. Thesedata indicate that PA treatment does in fact regulate T celldifferentiation.

FIG. 2 also shows data indicating that the E-03 dose of PA rectifies thedecrease in CD69−CD4+ observed in BXSB controls at early time points,although later losing this effect at longer time points of chronictreatment. In contrast, the E-07 dose appears to be effective at bothshort and long time points. These data are consistent with features ofthe Bio-Regulatory regimen: 1) The dose response curves are gaussian;and 2) the small amounts of PA that produce the effects suggest aprocess oriented target instead of a more traditional single effectortarget.

Table 25 shows the effect of chronic PA (1E-05 μG/dose) administeredthree times per week over a 6 and 9 week period compared with acutetreatment at the same amount three times per week for a single 3 weekperiod followed by an additional 6 and 9 weeks without treatment. Theacute treatment is effective in modulating the CD marker display.Although complex the data demonstrate the inter-relationship between thevarious markers; again the CD 8 series appear to be least sensitive toPA than the CD4 or B-cell series.

TABLE 25 Acute vs Chronic Treatment 0.00001 ug PA Percentage CD69+CD4−CD69+CD4+ CD4+CD69− CD4+CD8− CD4+CD8+ CD8+CD4− CD69+CD8− CD69+CD8+CD8+CD69− BXSB Control 19.73 12.45 67.50 41.85 8.48 49.24 21.53 7.8070.67 6 wks acute 56.25 22.42 21.34 17.64 7.68 73.59 24.91 5.56 69.54 9wks Acute 63.37 18.82 17.81 24.38 12.81 61.90 42.74 7.16 50.10 BL6Control 7.58 7.63 84.69 73.81 7.60 18.41 29.61 5.01 65.56 Rx Chronic 6wks 10.90 83.69 5.39 4.62 10.35 85.03 6.70 10.13 83.17 Rx Chronic 9 wks46.20 23.09 30.45 16.64 10.40 72.96 23.02 12.74 64.23

Table 26 shows a time profile of a single PA dose (1E-05 μG) on the nineT-cell marker series used in these studies. Again, the double positivecells (destined for apoptosis) as well as the activation series with CD8are relatively refractory to treatment. PA treatment does modulate theactivation CD4 series and mature cytotoxic T-cells (CD8+CD4−).

TABLE 26 Treatment with 0.00001 ug PA Percentage CD69+CD4− CD69+CD4+CD4+CD69− CD4+CD8− CD4+CD8+ CD8+CD4− CD69+CD8− CD69+CD8+ CD8+CD69−  3wks 29.79 12.41 57.8 42.57 7.22 50.21 24.98 4.78 70.24  6 wks 10.9 83.695.36 4.62 10.35 85.03 6.7 10.13 83.17  9 wks 46.2 23.09 30.45 16.64 10.472.96 23.02 12.74 64.23 12 wks 55.87 19.68 24.27 12.05 14.68 73.27 25.4417.5 57.06 Untreated BL 6 7.58 7.63 84.68 73.8 7.6 18.41 29.61 5.0165.55 Untreated BXSB 44.34 19.69 35.78 15.92 13.91 70.16 22.54 13.6963.76

In sum, the studies indicate that acute or chronic PA treatmentregulated T-cell CD markers in BXSB mice in a dose and kinetic dependentmanner, which correlate with the functional changes described abovewhich include partial restoration of normal mitogenic responses (Example5), reduction of autoantibody production (Example 6), and reduction ofcytotoxicity response (Example 7). Thus, PA regulates thedifferentiation sequence of cells of lymphoid lineage.

What is claimed is:
 1. A method for treating an immune dysfunction in asubject with an immune dysfunction, comprising administering to thesubject a composition comprising an amount of protein A (PA), whereinthe protein A (PA) consists of monomeric protein A (PA), sufficient totreat the immune dysfunction.
 2. The method of claim 1, wherein theimmune dysfunction comprises insulin-dependent diabetes mellitus.
 3. Amethod of treating existing tissue or cell damage or inhibiting tissueor cell damage in a subject caused by insulin-dependent diabetesmellitus, comprising administering to the subject a compositioncomprising an amount of protein A (PA), wherein the protein A (PA)consists of monomeric protein A (PA), sufficient to treat the existingtissue or cell damage or inhibit tissue or cell damage caused byinsulin-dependent diabetes mellitus.
 4. The method of claim 3, whereinthe tissue or cell damage is caused by a chronic or acute inflammatoryresponse or inflammation.
 5. The method of claim 4, wherein theinflammatory response or inflammation is at least in part mediated by anantibody or at least in part mediated by cellular immunity.
 6. Themethod of claim 1 or 3, wherein the composition comprises apharmaceutical formulation.
 7. The method of claim 1 or 3, wherein thesubject is human.
 8. The method of claim 1 or 3, wherein the monomericPA composition is administered systemically.
 9. The method of claim 1 or3, wherein the monomeric PA composition is administered locally.
 10. Themethod of claim 1 or 3, wherein the monomeric PA composition isadministered intravenously, orally, intramuscularly, intraperitoneally,intradermally, subcutaneously, intracavity, intracranial, transdermally,or parenterally
 11. The method of claim 1 or 3, wherein the monomeric PAcomposition is administered via a microencapsulated delivery system oran implant.
 12. The method of claim 1 or 3, wherein the monomeric PAcomposition is administered as a single or multiple dose over about 7 to45 days.
 13. The method of claim 1 or 3, wherein the monomeric PAcomposition is administered as a single or multiple dose over about 1 to15 weeks.
 14. The method of claim 1 or 3, further comprisingadministering a drug that reduces an inflammatory response orinflammation.